Abstract The goal of this application is to explore therapeutic opportunities for visceral pain in a new set of cellular targets within the spinal cord that will drive affective pain, rather than reflexive-defensive reactions. Pain from deep tissues (e.g. visceral organs, joints, muscles and bones) is among the most prevalent and disruptive. Major causes include inflammation, nerve injury and tumor growth. Translation of preclinical insight into the neurobiology of pain towards new medications has been disappointing. In preliminary studies, my colleagues and I have unmasked one of potential explanations for the lack of progress. Put briefly, in a series of studies on cutaneous pain, we have identified a group of spinal ascending projection neurons, marked by Tac1-Cre, that are essential for affective pain-indicative coping behaviors, but dispensable for first-line nocifensive-defensive reactions. This disconnect at the level of cutaneous pain leads to a testable hypothesis. We propose that affective and defensive elements of the response to noxious visceral stimuli could also be segregated at the level of the spinal cord. Our study plan uses innovative genetic tools to probe testable predictions of this hypothesis. We have two specific aims: Aim 1 is to test the prediction that unique spinal substrates are associated with acute visceromotor reflexes versus affective visceral pain. This prediction is based on our preliminary results, showing that two groups of spinal excitatory neurons (one is Tac1-Cre neurons), which are activated by noxious colorectal distensions (CRD), send extensive ascending projections to distinct, though partially overlapped sets of thalamic and midbrain nuclei. Meanwhile, we will test if a group of spinal inhibitory neurons activated by CRD act to gate visceral motor reflexes and/or affective pain. Aim 2 is to test the prediction that unique spinal substrates are associated with inflammatory visceral pain. Our pilot studies have identified two groups of spinal excitatory neurons that are activated by CRD only after gastrointestinal (GI) inflammation, and one of which (marked by VGLUT3-Cre) appears to be crucial for mediating sensitized affective visceral pain. Together with three other groups of excitatory neurons that are already activated by CRD under nave conditions, we will determine if these five groups of neurons transmit sensitized visceromotor reflexes, affective visceral pain, as well as the referred cutaneous pain induced by GI inflammation. All together, these studies will provide new insight into spinal substrates mediating different dimensions of acute and chronic visceral pain.